ExAC Database Research Articles

Report of a child with Bainbridge-Ropers syndrome due to a novel variant of ASXL3 gene and a literature review

To explore the clinical phenotype and genetic basis of a child with Bainbridge-Ropers syndrome (BRPS). A child with BRPS who had visited Nanjing Children's Hospital on June 26, 2019 was selected as the study subject. Clinical data of the child was reviewed. Genomic DNA was extracted from peripheral blood samples of the child and her parents. Whole exome sequencing (WES) was carried out, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The child was a 6-month-old girl with peculiar facial features, feeding difficulties, malnutrition, global developmental delay, hypotonia, mildly elevated aminotransferase and ulnar deviation. Results of WES showed that she has harbored a c.1533_1534del variant of the ASXL3 gene. Sanger sequencing confirmed that neither of her parents has carried the same variant. No similar case had been retrieved from the HGMD and ClinVar databases. No frequency for this variant among Asian populations was available in the ExAC, 1000 Genomes, and gnomAD databases. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.1533_1534del variant of the ASXL3 gene was determined to be likely pathogenic (PVS1+PS2+PM2_Supporting). The ASXL3 gene c.1533_1534del variant probably underlay the BRPS in this child. Above finding has provided a reference for the clinical diagnosis and genetic counseling for children with similar disorders.

Predisposing deleterious variants in the cancer-associated human kinases in the global populations.

Human kinases play essential and diverse roles in the cellular activities of maintaining homeostasis and growth. Genetic mutations in the genes encoding the kinases (or phosphotransferases) have been linked with various types of cancers. In this study, we cataloged mutations in 500 kinases genes in >65,000 individuals of global populations from the Human Genetic Diversity Project (HGDP) and ExAC databases, and assessed their potentially deleterious impact by using the in silico tools SIFT, Polyphen2, and CADD. The analysis highlighted 35 deleterious non-synonymous SNVs in the ExAC and 5 SNVs in the HGDP project. Notably, a higher number of deleterious mutations was observed in the Non-Finnish Europeans (26 SNVs), followed by the Africans (14 SNVs), East Asians (13 SNVs), and South Asians (12 SNVs). The gene set enrichment analysis highlighted NTRK1 and FGFR3 being most significantly enriched among the kinases. The gene expression analysis revealed over-expression of NTRK1 in liver cancer, whereas, FGFR3 was found over-expressed in lung, breast, and liver cancers compared to their expression in the respective normal tissues. Also, 13 potential drugs were identified that target the NTRK1 protein, whereas 6 potential drugs for the FGFR3 target were identified. Taken together, the study provides a framework for exploring the predisposing germline mutations in kinases to suggest the underlying pathogenic mechanisms in cancers. The potential drugs are also suggested for personalized cancer management.

SAT604 An Extensively Invasive Giant Prolactinoma Associated With CDH23 Mutation

Abstract Disclosure: B. Alghamdi: None. L.A. Alobaid: None. M. Alswailem: None. E.O. Othman: None. M. Dababo: None. O. Alsagheir: None. A.S. Alzahrani: None. Pituitary adenomas (PA), renamed recently pituitary neuroendocrine neoplasms, are common and prolactinomas are the most common subtype of these tumors. The vast majority of PA are sporadic but they can also be due to germline (∼ 5%), mosaic (<1%) or somatic mutations (∼40%). PA with germline mutations might be part of a syndrome such as MEN1 or apparently sporadic, either due to de novo mutations or because of low penetrance of the underlying gene (e.g. AIP). Among the several genes that have been identified, CDH23 was reported to be associated with familial PA in a single study that included 12 families with PA (33% of them had CDH23 variants) and 125 sporadic PA (15 (12%) of them harbored CDH23 variants) compared to only 0.8% of 260 healthy controls. In this report, we describe a middle-aged woman who was incidentally found to have a huge macroprolactinoma with extensive invasion. Genetic testing revealed a novel germline CDH23 variant. Case report: A 53-year-old woman who with hypertension, diabetes and chronic renal insufficiency diagnosed 4 years before. She accidentally fell down on the back of her head. A CT and subsequent MRI scans of the head showed a huge mass extensively involving the Sella, suprasellar space, the cavernous sinuses and middle fossae. This mass extends inferiorly to the sphenoid and parasellar sinuses, nasopharynx, and appears at the nostrils. The patient reported chronic headaches, snoring and congested nose, and worsening hearing and vision over the last 4 years. She denied history of galactorrhea and her periods ceased 8 years before. She never got married and has no children. Her family history is negative for any pituitary or other tumors. The mass was initially thought to be a nasopharyngeal cancer. However, a bedside biopsy from the protruding mass in the right nostril revealed a pituitary tissue strongly positive for prolactin and weakly positive for growth hormone (GH). An undiluted serum prolactin level was 47 ug/l (3.4-24). After several dilutions for the hook effect, the level was 578,000 ug/l (3.4-24). IGF-1 253 ng/ml (93-245), random GH 4.7 ng/ml, TSH 1.9 mU/l (0.4-4.2), FT4 11.4 pmol/l (12-22), AM cortisol 235 nmol/l (159-620), ACTH 27.6 (5-60). Whole exome sequencing followed by Sanger sequencing revealed a novel variant in CDH23 (c.2621C>A, p.Ala874Asp). Several in silico analyses suggest that this variant is pathogenic and it is assigned variant of unknown significance (VUS) according to American College of Medical Genetics (ACMG) classification. It is not reported in EXAC and 1000G databases. All other known PA-associated genes and cancer-associated genes revealed no pathogenic variants. Conclusion: We report a massive prolactinoma, likely due to a novel CDH23 variant. This is the second report on the likely pathogenic role of this gene in PA. In the case reported here, it is associated with a huge and highly invasive pituitary macroadenoma. Presentation: Saturday, June 17, 2023

Whole Exome Sequencing Reveals Two Novel Homozygous Variants in the TRAPPC9 and PLOD3 Genes Leading to Intellectual Disability and Bone Fragility with Contractures

Salt and Pepper’ syndrome is an autosomal recessive condition characterized by severe intellectual disability (ID), epilepsy, scoliosis, choreoathetosis, dysmorphic facial features, and altered dermal pigmentation. IDs are heterogeneous group of disorders prevalent in highly consanguineous populations and genetically variable condition of cognitive impairment linked with behavioral, syndromic, or dysmorphic features. Trafficking protein particle complex subunit 9 ( TRAPPC9) (MIM 611966) is an important gene, and mutations in this gene have been reported to be involved in ID conditions. Lysyl hydroxylase 3 (encoded by ( PLOD3)) is a multifunctional enzyme responsible for catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues, an important step during posttranslational modifications for collagen biosynthesis. In this study, whole exome sequencing was done for a family member having ID, microcephaly, included joint contractures, low bone mineral density, and dysmorphic feature. We identified a novel missense variant (NM_001160372.3) c.3211G>A, p.G1071S in the exon 22 of TRAPPC9 causing ID and other missense variant c.346G>A, p.V116M (NM_001084.4) in the exon 4 of the PLOD3 gene causing bone fragility with contractures in the affected member of the family. The obtained results were further validated by using Sanger sequencing analysis. The identified variant has not been reported in either gnomAD or Exac databases. Both parents were heterozygous (carriers) to these identified mutations. Our results for the first time suggest that identified novel TRAPPC9 and PLOD3 gene mutations are the main cause of the disease leading to the ID-associated conditions along with cognitive deficits along with microcephaly. This is the first study to report patients with TRAPPC9- and PLOD3-related disorder from Saudi Arabia. This study will add to the literature of the disease and will enable the creation of a database that will provide a strong base to understanding the critical genomic regions to control ID and related disorders in Saudi patients.

Clinical and genetic analysis of a very early-onset inflammatory bowel disease type 28 child with atypical clinical manifestation

To explore the clinical and genetic characteristics of a very early-onset inflammatory bowel disease (VEO-IBD) type 28 child with atypical clinical manifestations. A VEO-IBD type 28 child with atypical clinical manifestations admitted to the Department of Neonatology, Children's Hospital Affiliated to Shandong University on November 5, 2021 was selected as the study subject. Clinical data of the child was collected. Peripheral venous blood samples of the child and his parents were collected for high-throughput sequencing. Candidate variants were verified by Sanger sequencing and bioinformatic analysis. The child, a 50-day-old male, had manifested bronchitis, ulcerative stomatitis, eczema and slightly loose stool. High-throughput sequencing revealed that he has harbored compound heterozygous variants of the IL-10RA gene, namely c.299T>G (p.V100G) and c.301C>T (p.R101W), which were inherited from his father and mother, respectively. Bioinformatic analysis showed that both variants have been recorded in the HGMD database, though the c.299T>G variant has not been included in the gnomAD, 1000 Genomes, ExAC and ESP6500 databases, while the c.301C>T variant has a low population frequency. Both variants were predicted to be deleterious by the online software including SIFT, PolyPhen-2 and Mutation Taster. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be pathogenic (PS3+PM2_Supporting+PP3). The c.299T>G and c.301C>T variants of the IL-10RA gene probably underlay the VEO-IBD type 28 in this child. Above finding has expanded the phenotypic spectrum of VEO-IBD type 28 due to variants of the IL-10RA gene and provided a reference for the clinical diagnosis of this disease.

Clinical outcome and genotype analysis of four Chinese children with pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD) is a rare congenital hemolytic anemia. Here, wesummarized the clinical features and laboratory examinations of four Chinese children with PKD and analyze genomic mutations. Collected and analyzed the clinical data of all children and their parents and completed the relevant laboratory examinations of all children. Analyzed the sequences of related genes in children by second-generation sequencing technology and verified the suspected mutations in children's family by Sanger sequencing method or second-generation sequencing technology. A total of six mutations in gene PKLR were detected in four cases. Except for c.1510C>T (P1) and c.941T>C (P2 and P4), which had been reported in previous studies, the other four novel gene mutations were reported for the first time, including a rare homozygous mutation with large fragment deletion. All those gene mutations cause changes in the amino acids encoded by the gene, as well as subsequent changes in protein structure or loss of function. Compound heterozygous or homozygous mutations in the coding region of PKLR gene are the causes of PKD in these four Chinese children. The second-generation sequencing technology is an effective means to diagnose PKD. The mutations of c.457-c.462delATCGCC, c.1297T>C, c.1096C>T and Exon4-10del of PKLR reported in this article have not been included in the Thousand Genome Database, dbSNP(v138) and ExAC Database. The PKLR gene mutations found in these children with PKD can provide references for further research of the genetic characteristics of PKD and subsequent gene therapy.

Clinical and genetic analysis of three children with Menkes disease due to variants of ATP7A gene

To explore the clinical characteristics and genetic etiology of three children with Menkes disease. Three children who had presented at the Children's Medical Center, the Affiliated Hospital of Guangdong Medical University from January 2020 to July 2022 were selected as the study subjects. Clinical data of the children were reviewed. Genomic DNA was extracted from peripheral blood samples of the children, their parents and sister of child 1. Whole exome sequencing (WES) was carried out. Candidate variants were verified by Sanger sequencing, copy number variation sequencing (CNV-seq), and bioinformatic analysis. Child 1 was a 1-year-and-4-month male, and children 2 and 3 were monozygotic twin males aged 1-year-and-10-month. The clinical manifestations of the three children have included developmental delay and seizures. WES showed that child 1 has harbored a c.3294+1G>A variant of the ATP7A gene. Sanger sequencing confirmed that his parents and sister did not carry the same variant, suggesting that it was de novo. Children 2 and 3 had carried a c.77266650_77267178del copy number variation. CNV-seq results showed that their mother has carried the same variant. By searching the HGMD, OMIM and ClinVar databases, the c.3294+1G>A was known to be pathogenic. No carrier frequency has been recorded in the 1000 Genomes, ESP, ExAC and gnomAD databases. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the ATP7A gene c.3294+1G>A variant was predicted to be pathogenic. The c.77266650_77267178del variant has involved exons 8 to 9 of the ATP7A gene. ClinGen online system score for it was 1.8, which was also considered to be pathogenic. The c.3294+1G>A and c.77266650_ 77267178del variants of the ATP7A gene probably underlay the Menkes disease in the three children. Above finding has enriched the mutational spectrum of Menkes disease and provided a basis for clinical diagnosis and genetic counseling.

High carrier frequency of pathogenic PATL2 gene mutations predicted in population: a bioinformatics-based approach.

Topoisomerase II homologue 2 (PATL2) has been confirmed to be a key gene that contributes to oocyte maturation. However, the allele distribution and carrier frequency of these mutations remain uncharacterized. So a bioinformatics subcategory analysis of PATL2 mutations from outcome data and Single Nucleotide Polymorphism (SNP) databases was conducted. Altogether, the causative PATL2 mutation number detected in patients with oocyte maturation defects in the clinical studies and pathogenic PATL2 mutation sites predicted by software based on the database was approximately 53. The estimated carrier frequency of pathogenic mutation sites was at least 1.14‰ based on the gnomAD and ExAC database, which was approximately 1/877. The highest frequency of mutations detected in the independent patients was c.223-14_223-2del13. The carrier frequency of this mutation in the population was 0.25‰, which may be a potential threat to fertility. Estimated allele and carrier frequency are relatively higher than those predicted previously based on clinical ascertainment. A review of PATL2 mutation lineage identified in 34 patients showed that 53.81%, 9.22% and 14.72% of the oocytes with PATL2 mutations were arrested at the germinal vesicle (GV) stage, metaphase I (MI) stage and first polar body stage, respectively. Oocytes that could develop to the first polar body stage were extremely rare to fertilise, and their ultimate fate was early embryonic arrest. Phenotypic variability is related to the function of the regions and degree of loss of function of PATL2 protein. A 3D protein structure changes predicted by online tools, AlphaFold, showed aberrations at the mutation sites, which may explain partially the function loss. When the mutated and wild-type proteins are not in the same amino acid category, the protein structure will be considerably unstable. The integration of additional mutation sites with phenotypes is helpful in drawing a complete picture of the disease. Bioinformatics analysis of PATL2 mutations will help reveal molecular epidemiological characteristics and provide an important reference for new mutation assessment, genetic counselling and drug research.

Genetic analysis of a child with Kartagener syndrome due to novel compound heterozygous variants of DNAH5 gene

To explore the clinical characteristics and genetic basis of a child with Kartagener syndrome (KTS). Trio-whole exome sequencing was carried out for the child and his parents, and candidate variants were verified by Sanger sequencing. Changes in protein structure due to missense variants were simulated and analyzed, and the Human Splicing Finder 3.0 (HSF 3.0) online platform was used to predict the effect of the variant of the non-coding region. The child had featured bronchiectasis, sinusitis and visceral inversion. Genetic testing revealed that he has harbored compound heterozygous variants of the DNAH5 gene, namely c.5174T>C and c.7610-3T>G. Sanger sequencing confirmed the existence of the variants. The variants were not found in the dbSNP, 1000 Genomes, ExAC, ClinVar and HGMD databases. Protein structural analysis suggested that the c.5174T>C (p.Leu1725Pro) variant may affect the stability of local structure and its biological activity. The results of HSF 3.0 analysis suggested that the c.7610-3T>G variant has probably destroyed a splicing receptor to affect the transcription process. The compound heterozygous variants of the DNAH5 gene probably underlay the pathogenesis in the child. Above finding may facilitate the understanding of the clinical characteristics and genetic basis of KTS, and further expand the spectrum of DNAH5 gene variants.

Mutations in PLCZ1 induce male infertility associated with polyspermy and fertilization failure.

To investigate the genetic causes of polyspermy and total fertilization failure (TFF) in two independent male patients suffering from male infertility. Immunofluorescence (IF) staining was used to detect the localization of the PLCζ protein in sperm and the maternal pronucleus in the zygote. Genomic DNA samples were extracted from the peripheral blood of patients and their families. The ExAC database was used to identify the frequency of corresponding mutations. The PLCZ1 mutations were validated by Sanger sequencing. The pathogenicity of the identified mutations and their possible effects on the protein were assessed using in silico tools and molecular modeling. We identified a reported homozygous mutation c.588C > A (p.Cys196Ter) and a compound heterozygous mutation c.2T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in PLCZ1. The IF results showed that these multipronuclear zygotes formed as a result of polyspermy. In silico analysis predicted that the mutations result in disease-causing proteins. IF staining revealed that PLCζ is abnormally localized in the sperm samples from the two affected patients. Assisted oocyte activation (AOA) successfully rescued polyspermy and TFF and achieved pregnancy in two patients with the PLCZ1 mutation. We identified a homozygous mutation in PLCZ1 (c.588C > A [p.Cys196Ter]) in a male patient with polyspermy after in vitro fertilization (IVF) as well as a compound heterozygous mutation c.2T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in a male patient with fertilization failure after intracytoplasmic sperm injection (ICSI), and we provide evidence that the homozygous mutation can cause polyspermy and the compound heterozygous mutation can cause fertilization failure.

A novel homozygous mutation in TRAPPC9 gene causing autosomal recessive non-syndromic intellectual disability

BackgroundThe etiology of intellectual disabilities is diverse and includes both genetic and environmental factors. The genetic causes of intellectual disabilities range from chromosomal aberrations to single gene disorders. The TRAPPC9 gene has been reported to cause autosomal recessive forms of intellectual disabilities in 56 patients from consanguineous and non-consanguineous families around the world.MethodsWe analyzed two siblings with intellectual disability, microcephaly and delayed motor and speech development from a consanguineous Sudanese family. Genomic DNA was screened for mutations using NGS panel (NextSeq500 Illumina) testing 173 microcephaly associated genes in the Molecular Genetics service in Robert Debre hospital in Paris, France.ResultsA novel homozygous mutation (NM_031466.7 (TRAPPC9):c.2288dup, p. (Val764Glyfs*7) in exon 14 of TRAPPC9 gene was found in the two patients. The mutation was predicted to cause nonsense mediated decay (NSMD) using SIFT prediction tool. The variant has not been found in either gnomAD or Exac databases. Both parents were heterozygous (carriers) to the mutation.ConclusionThis is the first study to report patients with TRAPPC9-related disorder from Sub-Saharan Africa.

Abstract 11984: A Novel Pathogenic Gata6 Variant Identified in a Family With Persistent Truncus Arteriosus, Childhood-Onset Diabetes Mellitus and Spontaneous Intestinal Perforation

GATA6 is a member of the GATA family of transcription factors, among which GATA4, 5 and 6 are known to play critical roles during heart development. Pathogenic variants in GATA6 have been primarily associated with congenital heart disease (CHD) and pancreatic agenesis in humans. Although the phenotypic spectrum associated with pathologic variation in GATA6 has expanded to include developmental delay along with congenital malformations affecting the biliary system and diaphragm, the full disease spectrum of GATA6 pathogenic variation and genotype-phenotype correlations are unclear. Here, we report a family where the father had persistent truncus arteriosus (PTA) and three children had CHD, 2 with PTA and 1 with an atrial septal defect. Notably, the father had childhood-onset diabetes mellitus and one child had spontaneous intestinal perforation (SIP). Using exome sequencing, we identified a novel, heterozygous missense variant in GATA6 (c.1403G>A; p.Cys468Tyr) in affected members. This variant is absent from the gnomAD and ExAC databases and causes a missense change predicted to be damaging by all in silico bioinformatic tools. Sanger sequencing confirmed the exome sequencing results and segregation of this variant. GATA6 p.Cys468Tyr variant affects the highly conserved zinc-finger domain of GATA6 and impedes its ability to bind DNA. Using wild-type and mutant GATA6 constructs driving atrial natriuretic peptide promoter reporter, in vitro luciferase assays showed that p.Cys468Tyr mutant protein exhibited significantly decreased luciferase transcriptional activity. Furthermore, immunofluorescence revealed that p.Cys468Tyr mutant demonstrated an impaired localization pattern in the nucleus and induced protein aggregation. Our findings expand the variant and phenotypic spectrum for GATA6. Our study highlights that GATA6 variants can cause not only cardiac and pancreatic malformations but also gastrointestinal abnormalities, including neonatal SIP. Further investigation is needed to define the mechanisms underlying the full phenotypic spectrum associated with pathogenic variation in GATA6 .

Whole exome sequencing identified a novel POT1 variant as a candidate pathogenic allele underlying a Li-Fraumeni-like family.

Li-Fraumeni syndrome (LFS) and Li-Fraumeni-like (LFL) syndrome are rare hereditary diseases characterized by predisposition to a diverse spectrum of cancer types, primarily sarcoma. The pathogenic variants underlying the majority of LFL cases remain to be explored. We performed whole-exome sequencing (WES) on 13 core members of a large LFL family with highly aggregated incidences of cancers, including cases with sarcoma, non-small cell lung cancer and cardiac angiosarcoma, and conducted a comprehensive literature review of candidate gene associations in LFS/LFL syndromes or sarcoma to identify potential pathogenic germline variants. No germline variants in the best-known LFL/LFS-associated gene TP53 were detected. Of all the genes associated with LFS/LFL or sarcoma that we have surveyed, we identified a novel p.P35L germline variant in POT1 (protection of telomeres 1). Germline and somatic alterations in POT1 have been implicated in a series of familial cancers, including angiosarcoma, glioma, melanoma and colorectal cancer. This particular variant is located in the telomere-binding OB1 domain, which is important in maintaining the proper telomere length, and showed high conservation across different POT1 orthologues. No record of the variant was found in any of the 1000 genomes, ExAC, gnomAD, dpSNP and COSMIC databases. Prediction algorithms and in silico structural analysis suggested completely disrupted protein structure and function of POT1 in the presence of this mutation. Leveraging WES, we identified a novel germline risk allele, p.P35L in POT1, that likely predisposes to LFL syndrome. Our results support the routine testing of POT1 and other LFL/LFS-associated genes in the risk populations to enable early cancer diagnosis, prevention and intervention.

Novel mutations in TUBB8 and ZP3 cause human oocyte maturation arrest and female infertility

PurposeVariations in many genes may lead to the occurrence of oocyte maturation defectsand female infertility. The objective was to describe newly discovered mutations in TUBB8 and ZP3, and to characterise the accompanying spectrum of phenotypes and modes of inheritance. MethodsTUBB8 and ZP3 were sequenced from genomic DNA samples extracted from peripheral blood of patients and their family members by the whole-exome sequencing. The TUBB8 and ZP3 sequences are then aligned with cryptographic software to identify rare variations. Sanger sequencing and mass spectrometry were used to validate mutations. ExAC database was used to retrieve the frequency of corresponding mutations. PolyPhen-2 and PROVEAN were analyzed for mutations using silicon. ResultsWe identified Three novel mutations and two known variant in TUBB8 and ZP3 associated with maturation in five families, and fertilization and developmental arrest are in these patients. These mutations include four heterozygous mutations in TUBB8 (c.730G > A, p.Gly244Ser, c.124C > G, p.Leu42Val, c.1172G > T, p.Arg391Leu and c.178G > A, p.Val60Met), and a heterozygous mutation in ZP3 (c.400G > A, p.Ala134Thr). Among them, these variants of TUBB8 were highly conserved among primates. ConclusionAs far as we know, the TUBB8 mutations detected in our study at four sites have not been reported before, and the variant of ZP3 has been published as pathogenic. Our findings extend the known mutant spectrum of TUBB8 and ZP3, and provide insights into the etiology of infertility in human women. The exact molecular mechanism has not been analyzed and should be further investigated in the future.

Molecular analyses of metastatic collecting ducts renal cell carcinoma from the phase 2 prospective trial of cabozantinib as first-line treatment (BONSAI trial Meeturo 2).

e16507 Background: The phase II BONSAI trial ( n = 25 ; NCT 03354884) met the primary endpoint demonstrating activity of cabozantinib in untreated metastatic collecting ducts carcinoma (mCDC), a rare and biologically poorly characterized disease. Here we report on molecular analyses of baseline tissue samples. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 18 mCDC patients enrolled in BONSAI were sequenced by TruSeq RNA Exome kit (Illumina). The data were mapped and quantified using STAR and htseq, respectively. Globaltest and edgeR packages in R were used to assess the correlation between transcriptional profiles and survival data. Nineteen samples underwent DNA Sequencing with the Oncomine Comprehensive Assay Plus (ThermoFisher Scientific). The reads were aligned to the human genome reference (hg19) and analyzed with Opencravat and IonReporter software. Germline variants were excluded based on 1000 Genomes, GnomAd and Exac databases, and Clinical annotation of somatic variants was performed using ClinVar. Results: The global expression levels of thirty-one genes have been found significantly associated with overall survival (OS). The natural grouping of the 18 tumor samples based on the 31-gene signature identified a main group of 11 cases, showing global higher expression levels in 22 out of the 31 genes. This group displayed overall a significant higher OS rate in comparison to the remaining 7 patients, carrying opposite expression trend and mostly undergoing to disease progression. The identified signature was enriched in biological processes like cell junction, cytoskeleton organization and methylation. FOS oncogene was among the 9 genes negatively associated to OS, showing common higher expression in the poorer survival rate group. Furthermore, a 22-gene signature was found significantly associated with progression-free survival (PFS), involving mainly genes associated to cell cycle regulation or recognized as components of Golgi apparatus. Only three genes were globally up-regulated in the group of 9 patients characterized by shorter PFS. Finally, heterogeneous pattern of somatic mutations was identified in 19 tumor samples, with at least 8 genes recurrently affected in more than three patients. Notably, mutated genes were mostly involved in DNA repair and chromatin modification processes. Conclusions: Our findings for the first time define specific molecular signatures that differentiate therapy-specific outcomes in first-line mCDC, warranting further investigation of their involvement in the tumor biology. Clinical trial information: NCT 03354884.

Gene Spectrum and Clinical Traits of Nine Patients With Oocyte Maturation Arrest.

Background: Oocyte maturation arrest is a disease that produces immature oocytes and cannot be mature after culturing in vitro, which leads to female primary infertility. We aimed to summarize nine representative patients in our center to retrospectively analyze the genetic variants and clinical characteristics of oocyte maturation arrest. Methods: This study examined and analyzed nine families with oocyte maturation arrest. Whole-exome sequencing (WES) of the probands was performed to detect the pathogenic variants. Sanger sequencing verified the WES findings in patients and available parents. ExAC database was used to search the variant frequency. The variants were assessed by pathogenicity and conservational property prediction analysis and according to the American College of Medical Genetics and Genomics (ACMG). Phenotypes of oocytes were evaluated by a light microscopy, and the phenotype-genotype correlation was also evaluated. Results: Nine pathogenic variants in five genes were detected in nine patients, of which three were novel variants, including PATL2 [c.1374A > G (p. Ile458Met)] and [1289-1291del TCC (p. Leu430del)] and ZP2 [c.1543C > T (p. Pro515Ser)]. Nine variants were predicted to be pathogenic, resulting in different types of oocyte maturation arrest and clinical phenotypes. Conclusion: Three novel pathogenic variants were identified, enabling the expansion of the gene variant spectrum. The related pathogenic mutations of the PATL2, TUBB8, and ZP1∼3 genes were highly suggestive of being causative of oocyte maturation arrest.

In silico analysis of GATA4 variants demonstrates main contribution to congenital heart disease.

Introduction: Congenital heart disease (CHD) is the most common congenital abnormality and the main cause of infant mortality worldwide. Some of the mutations that occur in the GATA4 gene region may result in different types of CHD. Here, we report our in silico analysis of gene variants to determine the effects of the GATA4 gene on the development of CHD. Methods: Online 1000 Genomes Project, ExAC, gnomAD, GO-ESP, TOPMed, Iranome, GME, ClinVar, and HGMD databases were drawn upon to collect information on all the reported GATA4 variations.The functional importance of the genetic variants was assessed by using SIFT, MutationTaster, CADD,PolyPhen-2, PROVEAN, and GERP prediction tools. Thereafter, network analysis of the GATA4protein via STRING, normal/mutant protein structure prediction via HOPE and I-TASSER, and phylogenetic assessment of the GATA4 sequence alignment via ClustalW were performed. Results: The most frequent variant was c.874T>C (45.58%), which was reported in Germany.Ventricular septal defect was the most frequent type of CHD. Out of all the reported variants of GATA4,38 variants were pathogenic. A high level of pathogenicity was shown for p.Gly221Arg (CADD score=31), which was further analyzed. Conclusion: The GATA4 gene plays a significant role in CHD; we, therefore, suggest that it be accorded priority in CHD genetic screening.

Identification and characterization of genetic variants of TGFB1 in patients with congenital heart disease

BackgroundCongenital heart diseases (CHDs) are believed to be caused by abnormal gene functioning during embryonic heart development. Transforming growth factor-beta1 (TGFB1) is known to express in the early embryonic heart and regulates heart development. MethodsIn this study, the coding region of TGFB1 was screened for 238 CHD patients by Sanger sequencing. Case-control association study was performed to identify the risk allele for CHD. In silico and in vitro approaches were used to elucidate the role of rare missense variant of TGFB1 using P19 cell line. ResultsWe identified a rare missense variant (c.29C > G; p.P10R) in the signal peptide of the TGFB1 in two cases (MAF = 0.0042017), which was absent in 200 healthy controls. Although this variation is reported in the gnomAD (rs1800470, MAF =0.0002386) and the ExAC database (MAF = 0.00064), it is not reported in INDEX-db and GenomeAsia 100K databases. We also found three polymorphisms, namely c.29C > T; p.P10L, c.74G > C; p.R25P and c.788C > T; p.T263I. Case-control studies revealed that c.29C > T (rs1800470) variation is a risk factor, significantly associated with the CHD phenotype (OR = 1.4361, P = 0.0083). However, c.74G > C (rs1800471) and c.788C > T (rs1800472) alleles are not associated with the disease. Additionally, two rare synonymous variations, i.e. c.348C > T; p.T116T (MAF = 0.0042017) and c.501C > T; p.H167H (MAF = 0.00210084) were also identified in two and one cases, respectively. These were absent in the 200 controls. In silico analysis showed that missense variation p.P10R enhances the formation of the α-helix in the signal peptide, which possibly increases the TGFB1 secretion. The luciferase-reporter assay demonstrated significantly increased activity of p(SBE)4 (P = 0.016) and p(CAGA)12 (P = 0.0004) promoters in response to p.P10R mutant versus wild-type TGFB1. ConclusionThe p.P10R variant of TGFB1 implicated a gain-of-function activity which is potentially deleterious, while the c.29C > T variation is a risk factor associated with the CHD.

A novel RHCE*cE allele identified in a Chinese individual, encodes weakened expression of c and E antigens

A novel RHCE*cE allele identified in a Chinese individual, encodes weakened expression of c and E antigens

SYN1 Mutation Causes X-Linked Toothbrushing Epilepsy in a Chinese Family

Toothbrushing epilepsy is a rare form of reflex epilepsy (RE) with sporadic incidence. To characterize the genetic profile of reflex epilepsy patients with tooth brushing-induced seizures in a Chinese family. Solo clinical whole-exome sequencing (WES) of the proband, a 37-year-old Chinese man, was performed to characterize the genetic etiology of toothbrushing epilepsy. Mutations in the maternal X-linked synapsin 1 (SYN1) identified in the proband and his family members were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined using in silico analysis. The proband had four episodes of toothbrushing-induced seizures. The semiology included nausea, twitching of the right side of the mouth and face, followed by a generalized tonic-clonic seizure (GTCS). The proband's elder maternal uncle had three toothbrushing-induced epileptic seizures at the age of 26. The proband's younger maternal uncle had no history of epileptic seizures but had a learning disability and aggressive tendencies. We identified a deleterious nonsense mutation, c.1807C>T (p.Q603Ter), in exon 12 of the SYN1 gene (NM_006950), which can result in a truncated SYN1 phosphoprotein with altered flexibility and hydropathicity. This novel mutation has not been reported in the 1000G, EVS, ExAC, gnomAD, or HGMD databases. We identified a novel X-linked SYN1 exon 12 mutant gene in a Chinese family with toothbrushing epilepsy. Our findings provide novel insights into the mechanism of this complex form of reflex epilepsy that could potentially be applied in disease diagnosis.